Human chorionic gonadotropin (hCG) measurement is the basis of all pregnancy tests. hCG is produced by trophoblast cells of the placenta in pregnancy. It is also produced by trophoblast cells in gestational trophoblastic diseases (hydatidiform mole, quiescent gestational trophoblastic disease and choriocarcinoma) and in testicular and other germ cell malignancies. hCG is a glycoprotein composed of 2 dissimilar subunits, α- and β-subunit, coded by separate genes on separate chromosomes, held together by charge interactions. hCG α-subunit is composed of 92 amino acids and contains 2 N-linked oligosaccharides. hCG β-subunit is composed of 145 amino acids and contains 2 N-linked and 4 O-linked oligosaccharides. The 8 oligosaccharide side chains comprise >30% of the molecular weight of hCG, making it an exceptionally highly glycosylated glycoprotein (1-7).
hCG is a heterogeneous molecule. Peptide variants, cleaved or nicked forms of hCG, free subunits of hCG, and fragments of hCG are all detectable in serum and urine samples during pregnancy (1). Oligosaccharide variants reflect availability of sugar substrates, and general cellular metabolism (7-9), expression of different glycosyltransferases by cells (8,9). It has long been recognized that the hCG molecule, particularly the β-subunit of hCG, produced in choriocarcinoma (trophoblastic cancer) and testicular germ cell cancer migrates slower than hCG β-subunit standards on electrophoresis gels and elutes earlier than hCG β-subunit standards from gel filtration columns (10-12). Both of these findings indicate a larger molecular weight molecule. This has long been assumed to be due to the presence of large oligosaccharides on hCG β-subunit (10-12). Further studies with lectins and structural studies have indicated the presence of larger or more complex oligosaccharides on choriocarcinoma hCG (3,4,13). In 1987, there was demonstrated a major difference between the 4 O-linked oligosaccharides on hCG in choriocarcinoma and normal pregnancy hCG. The hCG from 10 normal pregnancies primarily contained a mixture of tri- and tetrasaccharides, with 13% hexasaccharide (range 0 to 14%). In contrast, choriocarcinoma hCG preparations contained over 50% of the hexasaccharide structure (4,5). This observation was confirmed one year later by Amano et al (6).
In 1997 it was shown that the difference in the 4 O-linked oligosaccharides is the principal variation between choriocarcinoma and pregnancy hCG. While first trimester normal pregnancy urine hCG contained 12.3 to 19% (mean=15.6%) hexasaccharide structures, choriocarcinoma urine hCG contained 48 to 100% (mean=74.2%) hexasaccharide structures (7). A smaller change was observed in α-subunit and β-subunit N-linked oligosaccharides (from an average of 6.8% and 14% triantennary structures in first trimester pregnancy to 9.8% and 51% triantennary structures in choriocarcinoma, on α- and β-subunit respectively (7)). We call the hCG produced in choriocarcinoma H-hCG because of the large size due to overly large sugar units (14,15). Using an individual choriocarcinoma preparation with 100% hexasaccharide type O-linked oligosaccharides (C5 hCG), we generated in collaboration with Birken and colleagues a H-hCG-specific antibody (antibody B152) (16), and established an immunoassay using the C5 hCG calibrated by amino acid analysis as standard (14,16,17). This assay specifically detect the hexasaccharide O-linked oligosaccharides on the C-terminal of choriocarcinoma H-hCG (18). In 1998 O'Connor et al. used the B152-based assay to show that H-hCG is the principal form of hCG made during early pregnancy, in the weeks following implantation (17). This finding has now been confirmed by these and other investigators (14, 15,19-22). It has also been shown that early pregnancy H-hCG is the same size as choriocarcinoma H-hCG (18)
Root trophoblast cells, or cytotrophoblasts, are mostly phenotypically invasive cells. These are the principal cells in choriocarcinoma tumors and in blastocysts at the time of implantation (20,23,24). While cytotrophoblasts produce H-hCG, differentiated syncytiotrophoblast cells produce regular hCG (14,20). As published previously (15), H-hCG and its free β-subunit account for all of the hCG immunoreactivity in the conditioned medium of JAR, JEG-3 and BeWo choriocarcinoma cell lines. Lectin Western blot studies indicate that these cell lines produce hCG with very similar oligosaccharide structures to C5 choriocarcinoma hCG (10,25).
A standard was needed for the antibody B152-base H-hCG assay, other than an individual urine H-hCG (patient C5). Culture fluid from JEG-3 cell line was selected for this purpose because H-hCG consistently accounted for ˜100% of the dimer immunoreactivity at 3 time points, reflecting sub-confluent and confluent culture densities and showing consistency with culture time (15). Large quantities of culture fluid were produced, and H-hCG was purified. The JEG-3H-hCG is used as standard for the commercial H-hCG test (invasive trophoblast antigen or H-HCG test, Nichols Institute Diagnostics, San Clemente Calif.). While this standard has not yet been calibrated against W.H.O. hCG standards, or formally adopted by W.H.O. it is the only standard available.
hCG's primary function in pregnancy is to maintain progesterone production by corpus luteal cells, however, H-hCG may have an independent function. As published, the total hCG immunoreactivity in the conditioned medium of JAR choriocarcinoma cells is H-hCG and its free β-subunit (15). Studies by Lei et al. (26), show that JAR cells are invasive in Matrigel membrane inserts in vitro, and are rapidly tumorigenic when transplanted into athymic nude mice in vivo. Lei et al. (26) treated JAR cells with hCG α-subunit antisense cDNA. This blocked secretion of the H-hCG and its free β-subunit. It also blocked Matrigel membrane insert invasion and tumorigenesis in athymic nude mice.